125:583Biointerfacial Characterization

Oct. 2 and 5, 2006Fluorescence Spectroscopy

Prof. Ed Castner

Chemistry Chemical Biology

Prof. Prabhas Moghe

Chemical & Biochemical Engineering

Introduction to Fluorescence

• Luminescence: emission of photons from electronically excited states of atoms, molecules, and ions.

• Fluorescence: Average lifetime from <10—10 to 10—7 sec from singlet states.

• Phosphorescence: Average lifetime from 10—5 to >10+3 sec from triplet excited states.

Reference Reading

• B. Valeur, “Molecular Fluorescence: Principles and Applications”, Chem. Library, call number: QD96.F56V35 2002

• J. Lakowicz, “Principles of Fluorescence Spectroscopy”, Chem. Library,call number: QD96.F56L34 1999

• W. Becker, “Advanced Time-Correlated Single-Photon Counting Techniques, Chem. Library,call number: QC793.5.P422B43 2005

Why Use Fluorescence Spectroscopy?

• Sensitivity to local electrical environment– polarity, hydrophobicity

• Track (bio-)chemical reactions

• Measure local friction (microviscosity)

• Track solvation dynamics

• Measure distances using molecular rulers: fluorescence resonance energy transfer (FRET)

Photophysics: Jablonski Diagram• Photoexcitation from the ground electronic

state S0 creates excited states S1, (S2, …, Sn)• Kasha’s rule: Rapid relaxation from excited

electronic and vibrational states precedes nearly all fluorescence emission.

– (track these processes using femtosecond spectroscopy)

• Internal Conversion: Molecules rapidly (10-14 to 10-11 s) relax to the lowest vibrational level of S1.

– (This is why DNA doesn’t emit much fluorescence)

• Intersystem crossing: Molecules in S1 state can also convert to first triplet state T1; emission from T1 is termed phosphorescence, shifting to longer wavelengths (lower energy) than fluorescence. Transition from S1 to T1 is called intersystem crossing. Heavy atoms such as Br, I, and metals promote ISC.

R =e−ΔE /kT

5

Fluorescence Probing: Solvation; Reorientation

Solvation Coordinate

)()0(

)()()(

∞−∞−

=νννν t

tC

hνlaser

Time-dependentfluorescenceStokes shift

)(2)(

)()()(

||

||

tItI

tItItr

⊥

⊥

+−

=

polarizationanisotropy

6

Fluorescence Lifetimes and Quantum Yields

Q =Γ

Γ + knr

• Quantum yield: ratio of the number of emitted photons to the number of absorbed photons.

• Fluorophores with highest quantum yields exhibit the brightest emission (e.g., rhodamines), when normalized to absorption strength.

• Γ is the fluorophore emission rate and the nonradiative decay to So rate is knr.

• The fluorescence quantum yield is given by

• Excited state lifetime: typically 10 ns,

Figure 1.13

τ =1

Γ + knr 7

Fluorescence Polarization Anisotropy

• Information about the size and shape of proteins or rigidity of various molecular environments.

• Fluorophores preferentially absorb photons whose electric vectors are aligned parallel with transition moment of the fluorophore. In an isotropic solution, fluorophores are oriented randomly. Upon excitation with polarized light, one selectively excites those fluorophore molecules whose absorption transition dipole is parallel to the electric vector of the excitation. This selective excitation results in partially oriented population of fluorophores and in partially polarized fluorescence emission.

• Fluorescence anisotropy r is defined by:• Polarization is defined by P:

– Where I|| and I are the fluorescence intensities of the vertically (||) and horizontally( ) polarized emission, when the sample is excited with vertically polarized light.

r =

IP −I⊥

IP + 2I⊥

P =

IP −I⊥

IP + I⊥

⊥⊥

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Rotational Dynamics: Anisotropy

•r(t) = distribution of relaxation times, relates to rotational diffusion•Fit equation with a multiple or a stretched exponential •Stretched Exponential Fit: r(t) = (r0-r)exp(-t/0) r

(above: Coumarin 343-/Na+ in 25% aqueous F88 triblock copolymer)

r(t)

0.4

0.3

0.2

0.1

0.0

252015105ns

0.100.050.00

-0.05-0.10

60

50

40

30

20

10

0

x103

403020100ns

III I

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Instrumentation: Time-Integrated Spectrofluorometer

10

Intrinsic Fluorophores

tetrapyrroles:hemeschlorophyllspheophytinscarotenoids

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Extrinsic fluorophores

• rhodamines

• fluoresceins

• coumarins

• carbocyanine dyes

• aromatic hydrocarbons and derivatives:– pyrenes, perylenes, anthracenes

• See Invitrogen Molecular Probes catalog

12

random coil(unimer)

micelles(above cmc/cmT)

hydrogels (above cgc/cgT)

Increasing Temperature (concentration)

R.K. Prud’homme et al Langmuir 1996 (12) 4651 (cubic gel structure)

Aggregate Structures in PEO-PPO-PEO Solutions

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Coumarin Fluorescence ProbesCoumarin Fluorescence Probes

Localizes in PPO hydrophobic/dry

core

Located primarily in wet phases

Localizes in PPO/PEO regions

(water?)

clogP = 4.08 clogP = 3.67 clogP = -1.09

C153 C102 C343-/Na+

N O O

CF3

ON

CH3

O N O O

O-/Na+

O

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Fluor. excitation and emission spectra

•Aq. PEO109-PPO41-PEO109

•5 w/v % solution forms micelles

•Probes localize in different regions

–Experience different electrical environments

N O O

CF3

ON

CH3

O

N O O

O-/Na+

O

15

C153C153

N O O

CF3

~17

nm

7.6-10.4 nm

N. J. Jain et al. JPCB 1998 (102), 8452.16

ON

CH3

O

C102C102

17

C343C343--/Na/Na++

N O O

O-/Na+

O

18

C343 — anion weakly sensitive to microphase transition

5 w/v% 25 w/v%

C153 and C102 — Blue Shift –Polar Non-polar

C102 — Blue shift at ~2-4 °C higher than C153•Distributed between PPO and the PEO-PPO interface

Temperature Dependent Emission Shifts

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Fluorescence Probing: ReorientationFluorescence Probing: Reorientation

)(2)(

)()()(

||

||

tItI

tItItr

⊥

⊥

+−

=

polarizationanisotropy

hνlase

r

Detection of emission de-polarization reports on micro-viscosity

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Simultaneously fit Anisotropy, r(t), double exponential reorientation 21

C153 Local friction (rot) increases by

3.5 times over the cmTExtremely sensitive to environmental changes in PPO core

C102 rot increases by ~ 2 times over cmTShifted to slightly higher TDistributed in multiple environments

C343-/Na+rot decrease scales roughly with decreasing macroscopic viscosity Mainly in bulk water/hydrated PEO regions

5 w/v% F88

25 w/v% F88

Grant, Steege, DeRitter, CastnerJ. Phys. Chem. B, 2005, 109, 22273. 22

C153 local friction increases from 14– 890 cp in gel forming concentration (25 w/v%)

63.08.34 η ×=NPol

rot

96.01.58 η ×=Pol

rot

Rheology estimates Tgel macroscopic viscosity ~107 cP

Calculated from Maroncelli et al J. Phys. Chem. A, 1997, (101) 1030 23

Principles of Time-Correlated Single-Photon Counting

(TCSPC)

see text by Wolfgang Becker,Chemistry Library

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